Exploring Fluorescence In-Situ Hybridization

In an attempt to improve sputum smear microscopy result readout, we are exploring fluorescence in-situ hybridization (FISH) technology as a strategy to improve upon identification and visualization of TB bacteria.

Sputum smear microscopy (SSM) remains the major diagnostic technique for tuberculosis (TB) detection in resource poor settings, despite several shortcomings associated with the methods used to visualize bacteria underneath a microscope.

Acid Fast Stain of bacteria (Mycobacterium bovis)

SSM typically utilizes a process referred to as Acid-Fast Staining (AFS). AFS is a chemical process that stains particular types of bacteria (TB) red against a blue background. While AFS has the ability to stain TB, it relies on both color and morphology (shape) of bacteria to determine the presence of TB. These parameters introduce ambiguity into the result readout, which contributes to the high rate of misdiagnosis of TB (≥50%). With SSM representing upwards of 90% of TB diagnosis in the developing world, improvement of result reliability is essential for addressing the global TB epidemic.

Diagram of FISH

In an attempt to improve SSM result readout, we are exploring fluorescence in-situ hybridization (FISH) technology as a strategy to improve upon identification and visualization of TB bacteria. FISH utilizes oligonucleotides probes that interact or “hybridize” with target genetic materials in the organism of interest. Once these probes hybridize, a fluorescent signal is produced, indicating that the genetic material present.

The FISH mechanism allows for potential SSM enhancement introducing a higher degree of sensitivity and specificity than AFS, as it neither relies on morphology or proper coloration of bacteria. These enhancements include the ability to determine strains of multi-drug resistant M.tb strains, a trait currently not possible with SSM diagnostics. Multi-drug resistant tuberculosis continues to be a growing problem with 480,000 reported cases in 2013, according to the World Health Organization’s 2014 Global tuberculosis report. This is further compounded by the fact that only 5% of people in the developing world with tuberculosis are screened for drug resistance.

FISH while powerful, is also cumbersome, time consuming and involves multiple steps. Early in-house research demonstrated that FISH could be successfully conducted on mycobacteria in 2-4 hours. Recent research at IV Lab has provided evidence that FISH could be conducted in fewer steps and in less time, increasing the feasibility of utilizing FISH for enhanced SSM in low-resource settings.

Check our blogs for updates about IV Lab’s discoveries in optimizing the FISH assay for use in conjunction with SSM to aid tuberculosis diagnostics in the developing world.